Affinity cell extraction by acoustics

ABSTRACT

Beads with functionalized material applied to them are exposed to an acoustic field to trap or pass the beads. The beads may include or be free of ferro magnetic material. The beads may be biocompatible or biodegradable for a host. The size of the beads may vary over a range, and/or be heterogenous or homogenous. The composition of the beads may include high, neutral or low acoustic contrast material. The chemistry of the functionalized material may be compatible with existing processes.

BACKGROUND

Separation of biomaterial has been applied in a variety of contexts. Forexample, separation techniques for separating proteins from otherbiomaterials are used in a number of analytical processes.

SUMMARY

Separation of biomaterials can be accomplished by functionalizedmaterial distributed in a fluid chamber that bind the specific targetmaterials such as recombinant proteins and monoclonal antibodies orcells. The functionalized material, such as microcarriers that arecoated with an affinity protein, is trapped by nodes and anti-nodes ofan acoustic standing wave. In this approach, the functionalized materialis trapped without contact (for example, using mechanical channels,conduits, tweezers, etc.).

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are described in more detail below, with reference to theaccompanying drawings, in which:

FIG. 1 is a diagram of a separation process using paramagnetic beads ina magnetic field;

FIG. 2 is a diagram of a separation process using acoustic beads in anacoustic field;

FIG. 3 is an image of CD3+ T-cell complexes with beads;

FIG. 4 is an image of beads without CD3− T-cells to demonstratespecificity of selection;

FIG. 5 is an image of heterogenous beads available with streptavidin andbiotin conjugates;

FIG. 6 is an image of homogenous agarose beads;

FIG. 7 is a photograph of a miniature acoustic system for processingbeads;

FIG. 8 is a photograph of a separation result;

FIG. 9 is a diagram of an affinity technique that may be used withbeads;

FIGS. 10, 11 and 12 are microphotographs of streptevidin-conjugated andbiotin-conjugated beads that form complexes with each other;

FIG. 13 is a microphotograph of a cell suspension with identification ofan Erythrocyte, a Dendritic cell and a T cell;

FIG. 14 is a microphotograph of a bright field image of cells;

FIG. 15 is a microphotograph of a bright field image of greenfluorescence of anti-CD4 antibodies bound to cells;

FIG. 16 is a microphotograph of a bright field image of cells;

FIG. 17 is a microphotograph of a bright field image of magentafluorescence of anti-CD4 antibodies bound to cells;

FIG. 18 is a series of microphotographs illustrate examples of bead-cellcomplexes in environments with an excess of beads and with an excess ofcells; and

FIG. 19 is a diagram of example activation chemistries for affinitybinding.

DETAILED DESCRIPTION

The affinity separation of biological materials, such as proteins orcells, is accomplished through the use of a ligand that is covalentlybonded to a surface, such as a microbead, interacts with the protein orcell such that the protein or cell is bound to the ligand on themicrobead.

A ligand is a substance that forms a complex with the biomolecules. Withprotein-ligand binding, the ligand is usually a molecule which producesa signal by binding to a site on a target protein the binding typicallyresults in a change of confirmation of target protein. The ligand can bea small molecule, ion, or protein which binds to the protein material.The relationship between ligand and binding partner is a function ofcharge, hydrophobicity, and molecular structure. Binding occurs byintermolecular forces such as ionic bonds, hydrogen bonds and van derWaals forces. The Association of docking is actually reversible throughdisassociation. Measurably irreversible covalent bonds between theligand and target molecule is a typical in biological systems.

A ligand that can bind to a receptor, alter the function of thereceptor, and trigger a physiological response is called an agonist forthe receptor. Agonist binding to receptor can be characterized both interms of how much physiological response can be triggered and in termsof the concentration of the agonist that is required to produce thephysiological response. High affinity ligand binding implies that therelatively low concentration of the ligand is adequate to maximallyoccupy a ligand-binding site and trigger a physiological response. Thelower the K_(i) level is, the more likely there will be a chemicalreaction between the pending and the receptive antigen. Low-affinitybinding (high K_(i) level) implies that a relatively high concentrationof the ligand is required before the binding site is maximally occupyand the maximum physiological response to the ligand is achieved.Bivalent ligands consist of two connected molecules as ligands, and areused in scientific research to detect receptor timers and to investigatethe properties.

The T cell receptor, or TCR, is a molecule found on the surface of Tcells or T lymphocytes, that is responsible for recognizing fragments ofantigen as peptides bound to major histocompatibility complex (MHC)molecules. The binding between TCR and antigen peptides is of relativelylow affinity and is degenerative.

Referring to FIG. 1, paramagnetic beads, such as iron or ferro-magneticbeads sold under the name Dynabeads, have been used to achieve affinityextraction. The magnetic beads, coated with a functionalized material,bind to biological targets in complex mixtures to permit the targetmaterial to be separated out of the complex mixture using a magneticfield. The beads carry molecules for affine binding various targets withhigh specificity. The beads are injected into the complex mixture andincubated to bind the targets. The beads are extracted by a magnettogether with the targets attached to the beads.

Micro sized beads are available, such as, e.g., Dynabeads, which are onthe order of 4.5 μm in size. Nano sized beads may be used, such as,e.g., Myltenyi, which are on the order of 50 nm in size. Some of theaffine molecules that may be used include antibodies, aptamers,oligonucleotides and receptors, among others. The targets for theaffinity binding may include biomolecules, cells, exosomes, drugs, etc.

Referring to FIG. 2, beads with high acoustic contrast and affinitychemistry are illustrated. These acoustic beads can be used in exactlythe same way as magnetic beads with regard to having functionalizedmaterial coatings or composition for affinity binding. The acousticbeads are designed to be extracted from a complex mixture or fluid withan acoustic field. The acoustic beads can be directly used in all theapplications developed in cell manufacturing, biochemistry, diagnostics,sensors, etc. that use magnetic beads.

The acoustic beads can use the same surface and affinity chemistry as isused with magnetic beads. This ease of substitution of acoustic beadsfor magnetic beads has many advantages, including simplifying approvalfor applications, as well as simplifying the applications.

The acoustic beads can be made biocompatible. Such beads can be producedin different sizes, which permits continuous separation based on size ina size differentiating acoustic field, such as may be provided with anangled-field fractionation technology. The acoustic beads can becombined with an enclosed acoustics-based system, leading to acontinuous end-to-end cycle for therapeutic cell manufacturing. Thisfunctionality provides an alternative to magnetic bead extraction, whilepreserving use of currently existing affinity chemistry, which can bedirectly transferred to the acoustic beads. The acoustic beads may be aconsumable product in the separation operation.

In an example, a proof of concept trial was made using the publishedMemorial Sloan Kettering Cancer Center (MSKCC) protocol for extractionof CD3+ T cells from patient's blood. In the trial, paramagnetic beadswere used, and the magnetic field is replaced with an acoustic field.The process of extracting CD3+ T cells from patient's blood is anintegral part of manufacturing CAR (chimeric antigen receptor) T cells.Current processes are based on commercially available CD3 Dynabeads. Inthe trial, efforts were made to minimize the protocol differences,including performing the experiments in culture broth, rather thanblood. The difference is considered reduced since several steps in CAR Tcell manufacturing work from broth. The solvent density was increased tomake T cells “acoustically invisible,” or not as susceptible to anacoustic field. The small size of the Dynabeads may provide an acousticcontrast that is similar to the cells, thus making separation tolerancessmaller. The trial employed Jurkat CD3+ and CD3− T cell lines as models.The CD3− cells were employed as a control for non-specific trapping.

Referring now to FIGS. 3 and 4, images of results of the trial areshown. The cell suspensions were incubated with CD3 Dynabeads, whichbound CD3+ cells. The mixture was passed through the acoustic system,which trapped the magnetic beads (with or without cells). The collectedcells were successfully growing in culture. The images in FIGS. 3 and 4are obtained with overlap of bright field images with fluorescenceimages. The beads are black with slight reddish autofluorescence. Thelive cells are fluorescent red. The bead diameter is 4.5 microns. FIG. 3shows CD3+ T-cell complexes with beads, which demonstrates theefficiency of the technique. FIG. 4 shows that no CD3− T-cells have beenextracted, which demonstrates the specificity and selectivity of thetechnique.

Referring now to FIGS. 5 and 6, results of a trial with acoustic beadsis shown. In this trial, agarose beads were used as the acoustic beads.These beads are available off-shelf from several manufacturers, and arenot paramagnetic or have little to none iron or ferro magnetic content.Some agarose beads have surface modifications that simplify antibodyattachment. They are also composed of biocompatible material, which canbe important for therapeutic solutions. FIG. 5 shows ABTBeads, which arerelatively inexpensive, heterogeneous (20-150 μm), off-shelf beads,which are available with streptavidin and biotin conjugates. FIG. 6shows CellMosaic agarose beads, which tend to be relatively expensive,homogeneous (20-40 μm), and can be configured with any modification byorder.

The acoustic beads can be trapped in an acoustic field, such as amulti-dimensional acoustic standing wave. Referring to FIG. 7, aminiature acoustic system developed for acoustic applications is shown,which is used for trapping the acoustic beads. The smaller size of thesystem contributes to reducing the need for larger amounts of expensivereagents and permits processing of small volume samples.

Referring to FIG. 8, CellMosaic agarose beads escaped (left tube) andtrapped (right) in the acoustic system are shown. The acoustic systemtrapping efficiency can be 90%+.

Referring to FIG. 9, a flexible approach to activating the acousticbeads is illustrated. In this approach, antibodies are attached toagarose beads using a streptavidin-biotin complex. The complex is widelyused in biochemistry, and very stable. Agarose beads with conjugatedstreptavidin are available commercially as are antibody-biotinconjugates.

The functionality of streptavidin-beads & biotin-beads was evaluated.Referring to FIGS. 10-12, streptevidin-conjugated and biotin-conjugatedbeads are shown to form complexes with each other, as expected, uponmixing,

It may be desirable to obtain independent isolation of CD4+ and CD8+(“helper” and “killer” T cells, respectively) from suspensions andmixing them in desired ratios with a view toward efficient therapy.Toward this end, acoustic beads with affinity for CD4 and CD8 receptorscan be provided. A trial to obtain an example was performed with cellsuspensions prepared from mice spleens. Referring to FIG. 13,identification of an Erythrocyte, a Dendritic cell and a T cell isprovided. About 20 million (M) and 18M CD4+ and CD8+ T-cells,respectively, have been isolated from 4 spleens using Invitrogendepletion kits. Both cell lines can grow, and both CD4 and CD8 T-cellsare about 8.2-8.6 μm.

In this trial, CD4+ and CD8+ isolated cells were verifiedimmunologically. Referring to FIGS. 14 and 15, verification of thepresence of CD4 receptors is obtained. Alexa488 anti-CD4 antibodies areused to estimate the amount of isolated CD4 T-cells after purificationfrom mouse spleens. FIG. 14 shows a bright field image with smallcircles being the cells in focal plane. FIG. 15 shows fluorescence ofanti-CD4 antibodies bound to the cells. FIG. 16 shows a bright fieldimage with small circles being the cells in focal plane. FIG. 17 showsfluorescence of anti-CD4 antibodies bound to the cells. The differentcolors of green and magenta in FIGS. 15 and 17, respectively, can allowmultiplex analysis of results, e.g., a CD4/CD8 ratio.

Referring now to FIG. 18, results of the trial are shown whereStreptavidin-conjugated agarose beads were employed withbiotin-conjugated anti-CD3 antibodies and CD3+ Jurkat T-cells. Theaffinity combinations of beads and cells is clearly illustrated. Thebeads can be separated out in an acoustic field to extract the cellsfrom the mixture.

Proof-of-concept and validation of performance has been shown usingacoustic affinity beads in an acoustic system. The disclosed methods andsystems permit the use of off-shelf reagents, and currently availableacoustic systems. The affinities can target any type of desired T cellsor markers including CD3+, CD4+, CD8+. The acoustic beads can have ahigh, neutral or low contrast factor, which can affect how the beadsrespond to an acoustic field, for example being urged toward an acousticnode or antinode, or passing through the field.

The beads may be composed of various materials and combinations, whichpermits development of optimal chemistry with acoustic performance andbiocompatibility. The beads may be processed for isolation, sorting orany other function useful in a separation process. When used with atuned acoustic system, the performance of specifically designed acousticbeads can match or exceed that of paramagnetic beads.

Existing chemistries may be used with the acoustic beads, and inconjunction with specifications of size and structure homogeneity toachieve desired results for acoustic and for isolation performance. Thebeads may be composed of composite constructs to advance acousticefficiency. The acoustic system provides flexibility to manage smallsizes, with heat management, and the use of fluidics to obtain resultsthat are not possible with paramagnetic beads alone. Thebiocompatibility and/or biodegradability of the acoustic beads andsimplified processing permits integration with existing hardware for CART cell manufacturing. The affinity acoustic beads can be used in anumber of environments, including model environments such as, e.g.,animal blood spiked with target cells and murine spleen extracts. Theacoustic beads may thus be used in collaboration with existing systems,and may be designed and manufactured for target applications. The beadsmay be provided with a core that is acoustically active or neutral, andthe bead themselves may be configured for high, neutral or low acousticcontrast. The size of the beads may be configured for separation andaffinity in combination, for example a certain sized bead may includefunctionalized material to target a certain biomaterial, while anothersized bead, may be functionalized to target another biomaterial, each ofwhich can be separated simultaneously and continuously in a closed orflowing system. The beads can be designed to be of a homogeneous sizedistribution within a narrow or relatively broad range. Various affinitychemistries may be used, including streptavidin-biotin complex andimmunoglobulin or aptamer. The beads may be designed for ease ofmanufacturability and/or for shelf-life. The beads may be used withapproved chemistries, so that they may readily be integrated into knownsystems that use approved chemistries.

Referring to FIG. 19, an illustration of example activation chemistriesis shown. The activation chemistries illustrated are applicable to theacoustic affinity beads described herein.

The methods, systems, and devices discussed above are examples. Variousconfigurations may omit, substitute, or add various procedures orcomponents as appropriate. For instance, in alternative configurations,the methods may be performed in an order different from that described,and that various steps may be added, omitted, or combined. Also,features described with respect to certain configurations may becombined in various other configurations. Different aspects and elementsof the configurations may be combined in a similar manner. Also,technology evolves and, thus, many of the elements are examples and donot limit the scope of the disclosure or claims.

Specific details are given in the description to provide a thoroughunderstanding of example configurations (including implementations).However, configurations may be practiced without these specific details.For example, well-known processes, structures, and techniques have beenshown without unnecessary detail to avoid obscuring the configurations.This description provides example configurations only, and does notlimit the scope, applicability, or configurations of the claims. Rather,the preceding description of the configurations provides a descriptionfor implementing described techniques. Various changes may be made inthe function and arrangement of elements without departing from thespirit or scope of the disclosure.

Also, configurations may be described as a process that is depicted as aflow diagram or block diagram. Although each may describe the operationsas a sequential process, many of the operations can be performed inparallel or concurrently. In addition, the order of the operations maybe rearranged. A process may have additional stages or functions notincluded in the figure.

Having described several example configurations, various modifications,alternative constructions, and equivalents may be used without departingfrom the spirit of the disclosure. For example, the above elements maybe components of a larger system, wherein other structures or processesmay take precedence over or otherwise modify the application of theinvention. Also, a number of operations may be undertaken before,during, or after the above elements are considered. Accordingly, theabove description does not bound the scope of the claims.

What is claimed is:
 1. A method for separating material in a fluid,comprising: applying a functionalized material to a plurality ofnon-magnetic biodegradable agarose beads that are acousticallyresponsive; in the fluid, exposing the beads to biomaterial with anaffinity for the functionalized material to permit the biomaterial tobind to the beads, wherein the biomaterial is T cells; and exposing thebeads in the fluid to an acoustic field and trapping the beads in theacoustic field.
 2. The method of claim 1, wherein the functionalizedmaterial is a streptavidin conjugate or a biotin conjugate.
 3. Themethod of claim 1, wherein the functionalized material is composed withan affinity for one or more of CD3, CD4 or CD8 receptors.
 4. The methodof claim 1, wherein the functionalized material includes affinemolecules that are one or more of antibodies, aptamers oroligonucleotides.
 5. The method of claim 1, wherein the functionalizedmaterial comprises a ligand.
 6. The method of claim 1, furthercomprising reversing a binding between the biomaterial and the beads. 7.The method of claim 1, further comprising: a first biomaterial and adistinct second biomaterial included in the biomaterial; isolating thefirst biomaterial and isolating the second biomaterial; and combiningthe first biomaterial and the second biomaterial in a predeterminedratio.
 8. The method of claim 1, further comprising: targeted materialand other material included in the biomaterial; and providing beads withan affinity for the targeted material to the fluid.
 9. The method ofclaim 8, further comprising increasing a density of the fluid to modifyan acoustic contrast between the T cells and the fluid.
 10. The methodof claim 1, wherein the plurality of beads include at least twodifferent sizes of beads, each size being configured with an affinityfor a different type of biomaterial.
 11. A method for separatingmaterial in a fluid, comprising: generating an acoustic standing wave ina chamber suitable for housing a fluid; providing non-magneticbead-biomaterial complexes to the chamber, wherein the beads areacoustically responsive non-magnetic biodegradable agarose beads, thebiomaterial is T cells and the T cells are bound to the beads via afunctionalized material with an affinity for the T cells; and in thefluid, trapping the non-magnetic bead-biomaterial complexes in theacoustic standing wave.
 12. The method of claim 11, wherein the acousticstanding wave is a multidimensional acoustic standing wave.
 13. Themethod of claim 11, further comprising binding the biomaterial to thenon-magnetic beads to form the bead-biomaterial complexes prior to beingprovided to the chamber.
 14. The method of claim 11, further comprisingtrapping greater than 90% of the non-magnetic bead-biomaterial complexesin the acoustic standing wave.